Molecular characterisation of multidrug-resistant Mycobacterium tuberculosis isolates from a high-burden tuberculosis state in Brazil.

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131 Mycobacterium tuberculosis (M.tb) DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of the katG, inhA and rpoB genes and RDRio sublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRio deletion was observed in 42 (32%) of the M.tb isolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%) M.tb isolates, we found mutations associated with rifampicin (RIF) resistance in rpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance in katG and inhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in the rpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area.

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Association of CYP7A1 -278A>C polymorphism and the response of plasma triglyceride after dietary intervention in dyslipidemic patients

We investigated the effect of the -278A>C polymorphism in the CYP7A1 gene on the response of plasma lipids to a reduced-fat diet for 6 to 8 weeks in a group of 82 dyslipidemic males with a mean age of 46.0 ± 11.7 years. Individuals who presented at least one high alteration in total cholesterol, low-density lipoprotein cholesterol or triglyceride values were considered to be dyslipidemic. Exclusion criteria were secondary dyslipidemia due to diabetes mellitus, renal, liver, or thyroid disease. None of the subjects were using lipid-lowering medication. Baseline and follow-up lipid concentrations were measured. The genotypes were determined by the digestion of PCR products with the BsaI restriction endonuclease. There were statistically significant reductions in plasma total cholesterol, low-density lipoprotein cholesterol and triglyceride concentrations after dietary intervention. The minor allele C has a frequency of 43 percent. Carriers of the C allele had significantly lower triglyceride concentrations (P = 0.02) than AA homozygotes. After adjustment of covariates, subjects with the AC and CC genotypes showed a greater reduction in triglyceride concentrations compared to subjects with the AA genotype. Multiple linear regression analyses showed that the AC and CC CYP7A1 genotypes accounted for 5.2 and 6.2 percent of triglyceride concentration during follow-up and adjusted percent of change of triglyceride concentration, respectively. The present study provides evidence that -278A>C polymorphism in the CYP7A1 gene can modify triglyceride concentrations in response to a reduced fat diet in a dyslipidemic male population. This gene represents a potential locus for a nutrigenetic directed approach.

Association of CYP7A1 -278A>C polymorphism and the response of plasma triglyceride after dietary intervention in dyslipidemic patients.

We investigated the effect of the -278A>C polymorphism in the CYP7A1 gene on the response of plasma lipids to a reduced-fat diet for 6 to 8 weeks in a group of 82 dyslipidemic males with a mean age of 46.0 +/- 11.7 years. Individuals who presented at least one high alteration in total cholesterol, low-density lipoprotein cholesterol or triglyceride values were considered to be dyslipidemic. Exclusion criteria were secondary dyslipidemia due to diabetes mellitus, renal, liver, or thyroid disease. None of the subjects were using lipid-lowering medication. Baseline and follow-up lipid concentrations were measured. The genotypes were determined by the digestion of PCR products with the BsaI restriction endonuclease. There were statistically significant reductions in plasma total cholesterol, low-density lipoprotein cholesterol and triglyceride concentrations after dietary intervention. The minor allele C has a frequency of 43%. Carriers of the C allele had significantly lower triglyceride concentrations (P = 0.02) than AA homozygotes. After adjustment of covariates, subjects with the AC and CC genotypes showed a greater reduction in triglyceride concentrations compared to subjects with the AA genotype. Multiple linear regression analyses showed that the AC and CC CYP7A1 genotypes accounted for 5.2 and 6.2% of triglyceride concentration during follow-up and adjusted percent of change of triglyceride concentration, respectively. The present study provides evidence that -278A>C polymorphism in the CYP7A1 gene can modify triglyceride concentrations in response to a reduced fat diet in a dyslipidemic male population. This gene represents a potential locus for a nutrigenetic directed approach.

Association of slow N-acetyltransferase 2 profile and anti-TB drug-induced hepatotoxicity in patients from Southern Brazil.

PURPOSE: To determine the frequency of N-acetyltransferase 2 (NAT2) polymorphisms, the NAT2 acetylation profile and its relation to the incidence of gastrointestinal adverse drug reactions (ADRs), anti-tuberculosis (TB) drug-induced hepatotoxicity, and the clinical risk factors for hepatotoxicity in a population from Brazil. METHODS: Two hundred and fifty-four Brazilian TB patients using isoniazid (INH), rifampicin (RMP), and pirazinamide (PZA) were tested in a prospective cohort study. NAT2 genotyping was performed by direct PCR sequencing. The association between gastrointestinal ADRs/hepatotoxicity and the NAT2 profile genotype was evaluated by univariate analysis and multiple logistic regression. RESULTS: Of the 254 patients analyzed, 69 (27.2%) were slow acetylators and 185 (72.8%) were fast acetylators. Sixty-five (25.6%) patients were human immunodeficiency virus (HIV)-positive. Thirty-three (13%) and 14 (5.5%) patients developed gastrointestinal ADR and hepatotoxicity, respectively. Of the 14 hepatotoxicity patients, nine (64.3%) were slow acetylators and five (35.7%) were fast acetylators. Sex, age, presence of hepatitis C virus, alcohol abuse, and baseline aminotransferases were not found to be risk factors for hepatotoxicity. However, logistic regression analysis revealed that slow acetylator status and the presence of HIV (p < 0.05) were independent risk factors for hepatotoxicity. CONCLUSIONS: Our findings show that HIV-positive patients that have the slow acetylation profile are significantly associated with a higher risk of developing hepatotoxicity due to anti-TB drugs.

Epidemiology of meningococcal disease in southern Brazil from 1995 to 2003, and molecular characterization of Neisseria meningitidis using multilocus sequence typing.

OBJECTIVE: To describe the epidemiology of meningococcal disease (MD) in southern Brazil. METHODS: Retrospective cohort study among 2215 MD cases reported from 1995 to 2003 in Rio Grande do Sul (RS) State. RESULTS: The overall incidence fell by 50%; the case-fatality rate during this period was 22%. Even so, the incidence of MD remained high after the epidemic period ended in 1999. Together, the age groups of 1-4 years and infants accounted for 54.1% of reported cases with incidences of 11.3/100 000 and 31.3/100 000, respectively; 69.8% of cases were caused by Neisseria meningitidis serogroup B, which increased significantly. There was a significant decrease in serogroup C cases in the whole period. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 caused almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by multilocus sequence typing. Twenty sequence types (STs) were identified, eight of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent; both belong to the ST-32/ET-5 complex. ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies in Brazil. CONCLUSION: Our data suggest that ST-33 and ST-259 clones and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS.

Pleural fluid ADA, IgA-ELISA and PCR sensitivities for the diagnosis of pleural tuberculosis.

The diagnosis of pleural tuberculosis (pTB) is difficult, and more sensitive and specific techniques are needed. In the period August 1998 to November 2002, we evaluated 132 patients with a pleural effusion submitted to a thoracentesis and pleural biopsy in a tertiary care hospital in Rio de Janeiro, Brazil. Three tests were performed and compared in the pleural fluid: ADA activity measurement, IgA-ELISA for two combined specific Mycobacterium tuberculosis antigens, and polymerase chain reaction (PCR) for detection of M. tuberculosis DNA. Ninety-five patients (72%) were given a final diagnosis of pTB. Overall histopathologic sensitivity was 77%. The sensitivities of pleural fluid culture and AFB smear were 42% and 1%, respectively. Twenty-one (22%) additional patients had a clinical diagnosis of pTB. Median follow-up time of all TB patients after the completion of antituberculous treatment was 13 months. Sensitivities of ADA, IgA-ELISA and PCR were 91%, 78% and 82%, while specificities were 93%, 96% and 85%, respectively. Only ADA sensitivity was significantly higher than the histopathologic examination (McNemar chi2 test; p = 0.002) and also significantly higher than ELISA (p = 0.049), but not higher than PCR (p = 0.143). We conclude that the routine use of ADA activity measurement in pleural fluid can obviate the need for a pleural biopsy in the initial diagnostic approach to pleural effusions, while IgA-ELISA and PCR techniques, potentially more specific tests, need further refinement to improve their accuracy.

Recent transmission of tuberculosis involving retired patients.

The reported incidence of tuberculosis (TB) in three different regions of Rio Grande do Sul State in Brazil varies considerably. We used IS6110-RFLP and spoligotyping methods to genotype Mycobacterium tuberculosis isolates obtained from 268 patients between 1998 and 2000 in order to assess the levels of recent transmission of TB in the three regions. The degree of clustering of the strain types did not differ among the three regions; neither did other characteristics such as demographic features, underlying medical conditions, or the proportion of resistant TB. As reported previously, male patients were at greater risk of developing TB and our data suggest that part of this may be related to the higher rates of recent transmission among them (P<0.05). In addition, we found that retired patients were almost 3 times more likely to be infected with cluster-pattern strains than patients reporting any other occupation (P<0.05), and more than 3 times more likely than non-retired patients in the same age group (P<0.05) to be infected with cluster-pattern strains. We conclude that recent transmission is not a major factor contributing to the differences in TB incidence in the three regions of Rio Grande do Sul. The reason for the suggested high proportion of recent transmission TB cases among the retired people needs further studies.

Low rate of occult hepatitis B virus infection among anti-HBc positive blood donors living in a low prevalence region in Brazil.

OBJECTIVE: The aim of this study was to determine the rate of occult hepatitis B virus (HBV) infection among blood donors living in a geographic region of low (5.6%) anti-HBc prevalence. SUBJECTS AND METHODS: Sera from 150 candidate blood donors whose blood was rejected due to total anti-HBc reactivity (despite absence of HBsAg) were tested for anti-HBs and IgM anti-HBc antibodies, as well as for HBeAg/anti-HBe. Serum HBV DNA was sought by using a PCR assay able to amplify part of the surface gene. Viral load was measured in the PCR positive samples. RESULTS: The pattern 'anti-HBc alone' (without HBsAg and anti-HBs antibodies) was found in 64 (42.7%) subjects. IgM anti-HBc and anti-HBe antibodies were detected in 2 (1.3%) and 80 (53.3%) samples, respectively. No sample was HBeAg-reactive. HBV DNA was repeatedly found in five (3.3%) samples, three of which were anti-HBs positive and two anti-HBs negative. All five HBV DNA positive samples showed a low viral load (<1000copies/ml). CONCLUSIONS: The data indicated a low rate of occult infection among anti-HBc positive, HBsAg negative blood donors living in a region of low prevalence of infection. Viral load was very low in all HBV infected subjects.

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory.

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x10(2) c.f.u. ml(-1). Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Aminophylline reversal of antihypertensive agent toxicity.

Hypotension occurred following a combined beta blocker (atenolol), angiotensin converting enzyme inhibitor (quinapil) and selective serotonin reuptake inhibitor (fluvoxamine) overdose. In another instance heart block and hypotension was noted in association with a diltiazem and atenolol adverse interaction. Crystalloid infusion was ineffective in both cases, but toxicity was rapidly reversed with aminophylline administration. Aminophylline's recognized inotropic and chronotropic properties make it a potentially valuable therapeutic agent in the treatment of antihypertensive medication toxicity.

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from Brazil.

Mutations in a 69-bp region of the rpoB gene associated with rifampin resistance (Rif(r)) in 100 isolates (82 Rif(r)) from three states of Brazil were studied. Twenty-one different kinds of mutations were identified in the Rif(r) isolates, and six new alleles are described.

Usefulness of IS6110-restriction fragment length polymorphism typing of Brazilian strains of Mycobacterium tuberculosis and comparison with an international fingerprint database.

Strains of Mycobacterium tuberculosis isolated from 219 different tuberculosis patients, 115 from patients residing in Rio de Janeiro, 79 from Rio Grande do Sul and the remaining from other regions of the country, were analyzed by IS6110-restriction fragment length polymorphism fingerprinting. The IS6110-DNA patterns from these strains were highly polymorphic: 174 different patterns were observed and 25 patterns were shared by 70 isolates (32%). Most strains (93.4%) had multicopy patterns and only 17% of clustered strains had less than six IS6110 copies. Strain clustering was significantly higher for isolates from Rio Grande do Sul (36.7%) in comparison with strains from Rio de Janeiro (22.6%), but only when using high stringency during cluster analysis. Upon screening of an international database containing 3,970 fingerprints of M. tuberculosis strains, 15% of the patterns of Brazilian strains (21% of the strains) were identical to a fingerprint of an isolate from another country and one particular eight-band pattern forming the largest Brazilian cluster was detected in seven additional countries, suggesting that international transmission of tuberculosis from and to Brazil could be occurring frequently. Alternatively,preferential use of certain IS6110 integration sites could also be important in high-copy number strains, having important consequences for the use of databases for epidemiological studies on a large scale.

Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli.

1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro.

Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli

1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro